Abstract: In order to further improve the culture efficiency and muscle quality of Monopterus albus,it is necessary to further explore the genes related to muscle development.In this study,three prokaryotic expression vectors of atrogin-1 were constructed:pCold-TF-atrogin-1,pET28a(+)-atrogin-1 and pGEX6P-1-atrogin-1,and the recombinant vectors were transferred into Transetta(DE3) competent cells. After induced expression of recombinant strains,only pCold-TF-atrogin-1/Transetta(DE3) strain expressed higher soluble recombinant protein.IPTG induction experiment showed that the concentration of inducing agent had little effect on protein solubility and content,and the best induction time was 20 h.The soluble fraction was purified by Ni-NTA column affinity chromatography and SDS-PAGE(10% polyacrylamide) electrophoresis confirmed the recombinant protein.The eukaryotic expression vector pEGFP-N1-atrogin-1 was constructed and the 293T cells were transfected.The experimental results showed that the atrogin-1 fusion protein was expressed in both the cytoplasm and the nucleus,and the expression level was relatively higher in the nucleus.

Key words: Monopterus albus, atrogin-1, Prokaryotic expression, Purification, Cell transfection, Cell localization

摘要： 为提高黄鳝养殖效率以及肌肉的质量，进一步发掘黄鳝肌肉发育相关基因，以atrogin-1为研究对象，构建了3种原核表达载体：pCold-TF-atrogin-1、pET28a (+)-atrogin-1及pGEX6P-1-atrogin-1。将构建的重组表达载体转入Transetta(DE3)感受态细胞，重组菌株经诱导表达后，仅pCold-TF-atrogin-1/Transetta(DE3)菌株表达出可溶性较高的重组蛋白。IPTG诱导试验结果表明，诱导剂浓度对蛋白质表达可溶性及含量几乎没有影响，诱导时间在20 h最佳。可溶性部分经Ni-NTA柱亲和层析纯化以及SDS-PAGE(10%聚丙烯酰胺)电泳，证实获得了重组蛋白。构建真核表达载体pEGFP-N1-atrogin-1，转染293T细胞，结果表明，atrogin-1融合蛋白在细胞质及细胞核中均有表达，且在细胞核中表达量相对较高。

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